Study On IL-13/p-IKKβ/NF-κB Signaling Pathway In Fibroblasts Co-cultured With Breast Cancer Cells

ObjectiveThe effect of IL-13 on the expression of p-IKK and NF-κB in fibroblast co-cultured with breast cancer cells was researched through establishing a co-culture model of human breast cancer cells with human fibroblasts in vitro and human breast cancer-burdened nude mice in vivo to simulate tumor microenvironment.This study tried to investigate the IL-13 signal pathway of fibroblast in breast tumor microenvironment and to provide experimental and theoretical basis for the mechanism of IL-13 effect on breast cancer matrix.Methods1.Cell co-culture in vitroBreast cancer cell line MDA-MB-231 was seeded in transwell and human embryonic skin fibroblast line ESF was inoculated on cell culture plates.These cells were co-cultured after adherence.ESF cells were collected for the subsequent experiments.2.Western blottingThe cells were washed twice with PBS before collecting.The RIPA lysis buffer was added at 4℃for 30min and cell suspension was centrifuged at 12000rpm for5min.Protein standards were prepared according to the instruction of BCA protein concentration assay kit.Colorimetric determination was performed using a microplate reader at 562nm,then a standard curve was drawn.The samples to be measured were placed in a 96 well plate and added with BCA working liquid,tested at 562nm by micro plate reader.The absorbance values of the wells were recorded.The corresponding protein content on the standard curve was found for the tested wells.Polyacrylamide gel electrophoresis（12%separated gel and 5%concentrated gel）was used.20μL samples were put in each lane.These samples were run in concentrated glue at 90V constant pressure for 25min and in separated glue at 120V constant pressure for about 90min.PVDF film were actived by 100%methanol for 15min.The protein of lanes was transfered to the film.The film was blocked by 5%skim milk powder at 4℃ on a shaker for 3h.The film was incubated by the primary antibody,which was diluted with 5%skim milk powder,at 4℃ overnight.The film was incubated for 2 h with secondary antibody after washed with TBST for 10 min total of3 times.ECL was used to develop the film.The optical density of lanes was analyzed by Image J software.3.Cell proliferation assay（CCK-8 method）Groups were formed according to the experiment.96-well plate was inoculated with a certain amount of cells.5 wells were set up in each group,and 100μL of cell suspension were placed in each well.10×CCK-8（10μL）were added for 2 h.Values were measured（at 450nm）every 24 h.4.Establishing an implanted model of human breast cancer in nude mice100μL cell suspension（2×10⁶MDA-MB-231 cells and 1×10⁶ESF cells）were injected into the breast of right chest in 6-7 week old female nude mice.The longest diameter and shortest diameters of tumor were measured with vernier caliper every other day.Tumor volume was calculated according to the formula V（mm³）=1/6πab2.Tumor diameter up to 5mm was consider tumorigenesis.When tumor diameter reached 7mm,IL-13 was injeted at multipoint of tumor tissues.Total concentration of IL-13 was 1μg/kg for each time.IL-13 was injected every other day,and total time was nine.After the 21st day of IL-13 intervene,the nude mice were killed by cervical vertebrae dislocation.The tumor tissues were fixed with 10%formalin.5.Immunofluorescence detectionTumor tissues of nude mice were embedded by paraffin,sliced,dewaxed and hydrated with gradient alcohol.Antigen was repaired with 0.01 mol/L citrate buffer（pH 6.0）,then washed with 0.01mol/L PBS（pH 7.4）3 times.After the sections were blocked with 1%BSA at 37℃ for 30min,the sections were incubated by the first antibody at 4℃ overnight.And the sections were incubated by the second antibody conjugated fluorescence at 37℃ for 60min.The results were examined by laser confocal microscope,photographed,and analyzed by Image-Pro Plus software.6.Immunohistochemistry experimentBasic steps were as follows.The sections were dewaxed with xylene and hydrated with gradient alcohol.Endogenous peroxidase was blocked by 3%H₂O₂.Antigen was repaired with 0.01 mol/L citrate buffer in microwave oven.Non-specific antigens of sections were closed by normal sheep serum at 37℃ for 20min.The first antibodies were added to sections in wet box at 4℃ overnight,then the corresponding secondary antibodies were added at room temperature for 60min.The sections were colorated by DAB,redyed by hematoxylin,differentiated by 1%HCL-alcohol for 10s,returned to blue by 1%ammonia,dehydrated by gradient alcohol,made transparent by xylene,then sealed by neutral resin.Finally,the sections were observed and photographed with microscope.These pictures were analyzed.7.Statistical analysisThe experimental results were expressed by mean±SD.The experimental data were processed by SPSS Statistics 17.0 software.t-test was used to compare the means between two groups.P<0.05 represented significant difference.P>0.05represented no significant difference.Results1.Experimental results in vitro（1）IKKβphosphorylation induced by IL-13 in co-cultured ESF cellsThe results of western blotting showed that p-IKKβexpression of ESF cells was higher in ESF+MDA-MB-231+IL-13 group than in ESF+MDA-MB-231 group（P<0.05）,and p-IKKβexpression of ESF cells was lower in ESF group than in ESF+MDA-MB-231+IL-13 group or ESF+MDA-MB-231 group（P<0.05）,which indicated that IL-13 could promote IKKβphosphorylation of co-cultured ESF cells.No significant difference was found in p-IKKβprotein expression between MDA-MB-231 group and MDA-MB-231+IL-13 group（P>0.05）,which indicated that IL-13 had no significant effect on p-IKKβof MDA-MB-231 breast cence cells under the condition of no stromal fibroblasts.（2）NF-κBp65 and NF-κBp50 expression up-regulated by IL-13 in co-cultured ESF cellsThe results of western blotting showed that NF-κBp65 and NF-κBp50 expression of ESF cells was higher in ESF+MDA-MB-231+IL-13 group than in ESF+MDA-MB-231 group（P<0.05）,and NF-κBp65 and NF-κBp50 expression of ESF cells was lower in ESF group than in ESF+MDA-MB-231+IL-13 group and ESF+MDA-MB-231 group（P<0.05）,which indicated that IKKβphosphorylation induced by IL-13 made contribution to downstream factors NF-κBp65 and NF-κBp50and that was what the NF-κBp65 and NF-κBp50 expression was up-regulated in co-cultured ESF cells.No significant difference was found in NF-κBp65and NF-κBp50 protein expression between MDA-MB-231 group and MDA-MB-231+IL-13 group（P>0.05）,which indicated that IL-13 had no significant effect on NF-κBp65 and NF-κBp50 of MDA-MB-231 breast cence cells under the condition of no stromal fibroblasts.（3）IL-13 and fibroblasts synergistically promote the proliferation of breast cancer cellsThe results of CCK-8 experiments showed that the proliferation of MDA-MB-231 cells in ESF+MDA-MB-231+IL-13 group was significantly higher than that in other groups from day 3 to day 7 of the culture（P<0.05）.The proliferation of MDA-MB-231 cells in ESF+MDA-MB-231 group was significantly higher than that in MDA-MB-231 group and MDA-MB-231+IL-13 group from day 4 to day 7 of the culture（P<0.05）.The results indicate that IL-13 acts on fibroblasts and the co-cultured microenvironment and IL-13 synergistically promote the proliferation of MDA-MB-231 cells.2.Experimental results in vivo（1）The results of HE staining transplantation tumor tissues of human breast cancer-burdened nude miceCancer tissues were confirmed by HE staining in transplantation tumor tissues.（2）The intervene of IL-13 up-regulated IKKβphosphorylation of human breast cancer xenografts in nude micep-IKKβwas detected by immunofluorescence and confocal microscopy.Green fluorescent flag was p-IKKβin transplantation tumor tissues.The green fluorescent intensity of p-IKKβwas detected by Image-Pro Plus software.The fluorescent intensity of p-IKKβwas significantly higher in ESF+MDA-MB-231+IL-13 group than in other groups（P<0.05）,which showed that the intervene of IL-13 could up-regulate IKKβphosphorylation in human breast cancer xenografts of the co-growth of ESF with MDA-MB-231 cells.No significant difference was found in fluorescent intensity of p-IKKβbetween MDA-MB-231+IL-13 and MDA-MB-231groups（P>0.05）,which indicated that tumor stromal microenviroment had an important effect on IL-13 action.（3）The intervene of IL-13 up-regulated NF-κBp65 and NF-κBp50 of human breast cancer xenografts in nude miceThe expression of NF-κBp65 and NF-κBp50 was detected by immunofluorescence and confocal microscopy.Green fluorescent flag was NF-κBp65and NF-κBp50 in transplantation tumor tissues.The green fluorescent intensity of NF-κBp65 and NF-κBp50 was detected by Image-Pro Plus software,and was significantly higher in ESF+MDA-MB-231+IL-13 group than other groups（P<0.05）,indicating that IKKβphosphorylation induced by IL-13 could target downstream factors NF-κBp65 and NF-κBp50 and up-regulated NF-κBp65 and NF-κBp50expression.No significant difference was found in fluorescent intensity of NF-κBp65and NF-κBp50 between MDA-MB-231+IL-13 and MDA-MB-231 groups（P>0.05）,indicating there was a close correlation between IL-13 action and tumor stromal microenvironment.3.Immunohistochemical results of clinic breast cancer tissuesFirstly breast cancer tissues were confirmed by HE staining,then detected by immunohistochemistry.The color was developed by DAB,and brown was positive expression.（1）IL-13 expressed in clinic breast cancer tissuesImmunohistochemical results showed that IL-13 was positive in human breast cancer tissues,and mainly distributed in cytoplasm.The results of optical density analyzed by Image-Pro Plus sofetware showed that the expression of IL-13 was higher in breast cancer tissues than that in breast non-cancer tissues（P<0.05）,indicating that IL-13 had a role in breast cancer occurrence and development.（2）p-IKKβwas positive in clinic breast cancer tissuesThe results of immunohistochemistry showed that p-IKKβwas positive in clinic breast cancer tissues,and mainly distributed in cytoplasm.The results of optical density analyzed by Image-Pro Plus sofetware showed that p-IKKβwas higher in human breast cancer group than human breast non-cancer group,which indicated that microenviroment of human breast cancer tissues could induce IKKβphosphorylation.Phosphorylated IKKβeffected on cancer tissues through its signal pathway.（3）NF-κBp65 and NF-κBp50 expressed in clinic breast cancer tissuesImmunohistochemical results showed that NF-κBp65 and NF-κBp50 were positive in human breast cancer tissues,and mainly distributed in cytoplasm and nuclei.The results of optical density analyzed by Image-Pro Plus sofetware showed that the expression of NF-κBp65 and NF-κBp50 was higher in human breast cancer group than human breast non-cancer group（P<0.05）,indicating that phosphorylated IKKβof human breast cancer tissues could activate molecules NF-κBp65 and NF-κBp50 of its signal pathway.Conclusions1.Under the condition of the co-culture with human breast cancer cells,IL-13 could up-regulate the expression of p-IKKβ,NF-κBp65 and NF-κBp50 of human fibroblasts.2.IL-13 up-regulates the expression of p-IKKβ,NF-κBp65 and NF-κBp50 of transplanted tumor tissues in human breast cancer-burdened nude mice.3.IL-13 and fibroblasts synergistically promote the proliferation of breast cancer cells4.IL-13,p-IKKβ,NF-κBp65 and NF-κBp50 highly express in clinic breast cancer tissues.